The invention relates generally to the field of nucleic acid sequence analysis. In certain embodiments, the analysis is genotyping. In certain embodiments, the analysis involves detecting single nucleotide polymorphisms (SNPs). The invention also relates to methods, kits, and computer software for nucleic acid analysis.
There are various methods for nucleic acid analysis. One method involves the use of restriction fragment length polymorphisms (RFLPs). Another method uses amplified fragment length polymorphisms (AFLPs).
Analysis of allelic differences between individuals is one area of nucleic acid analysis. In certain applications, one detects single nucleotide polymorphisms (SNPs). Approximately 2.9xc3x97106 SNPs have been mapped and sequenced in the human genome, which covers approximately 3xc3x97109 base pairs. Effective analysis of an individual""s genome on efficient and rapid scale would be very beneficial.
In certain embodiments, a method of nucleic acid analysis is provided. Certain such embodiments comprise generating multiple fragments of nucleic acid target sequence by digesting the target sequence with at least one restriction enzyme, and amplifying the multiple fragments employing a first set of primers to generate multiple amplified fragments.
According to certain embodiments, the method further comprises detecting a nucleotide for at least one of the amplified subsets of the multiple amplified fragments. According to certain embodiments, the amplifying includes using a second set of primers, which include at least one additional nucleotide on the 3xe2x80x2 ends of the first set of primers, to amplify a subset of the multiple amplified fragments to generate an amplified subset of the multiple amplified fragments.
In certain embodiments, the amplifying involves the use of one or more subsequent sets of primers, which include at least one additional nucleotide on the 3xe2x80x2 ends of the first set of primers, to sequentially create one or more subsets of amplified fragments.
In certain embodiments, the amplified subset of the multiple amplified fragments are asymmetrically amplified to generate single-stranded DNA prior to the detecting a nucleotide for at least one of the amplified subsets of the multiple amplified fragments.
According to certain embodiments, methods of performing single base extension reactions isothermally are provided.
According to certain embodiments, single stranded template is prepared for use in single base extension reactions. In certain embodiments, the single stranded template is prepared by asymmetric PCR.
According to certain embodiments, further amplification is performed with one primer from the first primer pair, and at least one primer which comprises a sequence corresponding to an area immediately adjacent to a nucleotide to be detected. According to certain embodiments, an excess of the primer from the first primer pair is added to generate a long oligonucleotide primer of a known length, wherein the 3xe2x80x2 end of the long primer is proximal to the nucleotide to be detected. In certain of these embodiments, labeled sequence terminators are added to the reaction containers, and a single base extension, or minisequencing, reaction is performed. In certain embodiments, the products of the minisequencing reactions are resolved according to the length of the long oligonucleotide. In certain embodiments, the minisequencing reaction is performed isothermally.
According to certain embodiments of the invention, a kit is provided. In certain embodiments, such a kit comprises a first pair of primers for the amplification of AFLPs. The first pair of primers comprise a first primer and a second primer. Such a kit further comprises a second set of primers, wherein the primers of the second set comprise a sequence identical to the primers used to generate the AFLPs, and further comprise a single nucleotide added to the 3xe2x80x2 end of the primer of the first primer set.
According to certain embodiments, software is provided. In certain embodiments this software identifies known SNPs from an electronic database, designs primers comprising sequence of an area immediately adjacent to a known SNP, calculates melting temperatures for the primers, and selects a primer based on the calculated melting temperature.